Publications

• Cloning and sequencing of four structural genes for the Na(+)-translocating NADH-ubiquinone oxidoreductase of Vibrio alginolyticus.

  Beattie P., Tan K., Bourne R.M., Leach D.R.F., Rich P.R., Ward F.B.

  Oligonucleotide probes based on the N-terminal amino acid sequences of the NqrA and NqrC subunits were used to clone genes for the Na(+)-dependent NADH-ubiquinone oxidoreductase complex from Vibrio alginolyticus. Four consecutive ORFs were identified encoding subunit proteins of 48.6, 46.8, 27.7 a ... >> More

  Oligonucleotide probes based on the N-terminal amino acid sequences of the NqrA and NqrC subunits were used to clone genes for the Na(+)-dependent NADH-ubiquinone oxidoreductase complex from Vibrio alginolyticus. Four consecutive ORFs were identified encoding subunit proteins of 48.6, 46.8, 27.7 and 22.6 kDa, respectively (NqrA-D). A further ORF, showing 71% homology to the BolA protein of Escherichia coli, was located upstream. From sequence comparisons, we conclude that the Na(+)-dependent NADH-ubiquinone oxidoreductase complex of V. alginolyticus is clearly distinct from the corresponding H(+)-dependent enzymes of both prokaryotes and eukaryotes. << Less

  FEBS Lett. 356:333-338(1994) [PubMed] [EuropePMC]

• Mutagenesis study of the 2Fe-2S center and the FAD binding site of the Na(+)-translocating NADH:ubiquinone oxidoreductase from Vibrio cholerae.

  Barquera B., Nilges M.J., Morgan J.E., Ramirez-Silva L., Zhou W., Gennis R.B.

  Many marine and pathogenic bacteria have a unique sodium-translocating NADH:ubiquinone oxidoreductase (Na(+)-NQR), which generates an electrochemical Na(+) gradient during aerobic respiration. Na(+)-NQR consists of six subunits (NqrA-F) and contains five known redox cofactors: two covalently bound ... >> More

  Many marine and pathogenic bacteria have a unique sodium-translocating NADH:ubiquinone oxidoreductase (Na(+)-NQR), which generates an electrochemical Na(+) gradient during aerobic respiration. Na(+)-NQR consists of six subunits (NqrA-F) and contains five known redox cofactors: two covalently bound FMNs, one noncovalently bound FAD, one riboflavin, and one 2Fe-2S center. A stable neutral flavin-semiquinone radical is observed in the air-oxidized enzyme, while the NADH- or dithionite-reduced enzyme exhibits a stable anionic flavin-semiquinone radical. The NqrF subunit has been implicated in binding of both the 2Fe-2S cluster and the FAD. Four conserved cysteines (C70, C76, C79, and C111) in NqrF match the canonical 2Fe-2S motif, and three conserved residues (R210, Y212, S246) have been predicted to be part of a flavin binding domain. In this work, these two motifs have been altered by site-directed mutagenesis of individual residues and are confirmed to be essential for binding, respectively, the 2Fe-2S cluster and FAD. EPR spectra of the FAD-deficient mutants in the oxidized and reduced forms exhibit neutral and anionic flavo-semiquinone radical signals, respectively, demonstrating that the FAD in NqrF is not the source of either radical signal. In both the FAD and 2Fe-2S center mutants the line widths of the neutral and anionic flavo-semiquinone EPR signals are unchanged from the wild-type enzyme, indicating that neither of these centers is nearby or coupled to the radicals. Measurements of steady-state turnover using NADH, Q-1, and the artificial electron acceptor ferricyanide strongly support an electron transport pathway model in which the noncovalently bound FAD in the NqrF subunit is the initial electron acceptor and electrons then flow to the 2Fe-2S center. << Less

  Biochemistry 43:12322-12330(2004) [PubMed] [EuropePMC]

• Sodium-dependent steps in the redox reactions of the Na+-motive NADH:quinone oxidoreductase from Vibrio harveyi.

  Bogachev A.V., Bertsova Y.V., Barquera B., Verkhovsky M.I.

  The Na+-translocating NADH:ubiquinone oxidoreductase (Na+-NQR) from Vibrio harveyi was purified and studied by EPR and visible spectroscopy. Two EPR signals in the NADH-reduced enzyme were detected: one, a radical signal, and the other a line around g = 1.94, which is typical for a [2Fe-2S] cluste ... >> More

  The Na+-translocating NADH:ubiquinone oxidoreductase (Na+-NQR) from Vibrio harveyi was purified and studied by EPR and visible spectroscopy. Two EPR signals in the NADH-reduced enzyme were detected: one, a radical signal, and the other a line around g = 1.94, which is typical for a [2Fe-2S] cluster. An E(m) of -267 mV was found for the Fe-S cluster (n = 1), independent of sodium concentration. The spin concentration of the radical in the enzyme was approximately the same under a variety of redox conditions. The time course of Na+-NQR reduction by NADH indicated the presence of at least two different flavin species. Reduction of the first species (most likely, a FAD near the NADH dehydrogenase site) was very rapid in both the presence and absence of sodium. Reduction of the second flavin species (presumably, covalently bound FMN) was slower and strongly dependent on sodium concentration, with an apparent activation constant for Na+ of approximately 3.4 mM. This is very similar to the Km for Na+ in the steady-state quinone reductase reaction catalyzed by this enzyme. These data led us to conclude that the sodium-dependent step within the Na+-NQR is located between the noncovalently bound FAD and the covalently bound FMN. << Less

  Biochemistry 40:7318-7323(2001) [PubMed] [EuropePMC]

• Identification of six subunits constituting Na+-translocating NADH-quinone reductase from the marine Vibrio alginolyticus.

  Nakayama Y., Hayashi M., Unemoto T.

  We previously reported that the purified Na+-translocating NADH-quinone reductase (NQR) from the marine Vibrio alginolyticus is composed of three major subunits, alpha, beta and gamma. NQR operon was sequenced and was found to be composed of 6 structural genes. Among these genes, nqr1, nqr3 and nq ... >> More

  We previously reported that the purified Na+-translocating NADH-quinone reductase (NQR) from the marine Vibrio alginolyticus is composed of three major subunits, alpha, beta and gamma. NQR operon was sequenced and was found to be composed of 6 structural genes. Among these genes, nqr1, nqr3 and nqr6 were identified to code for alpha-, gamma- and beta-subunits, respectively. The protein products from nqr2, nqr4 and nqr5, however, were not reported. The sequence data predicted that these three proteins are very hydrophobic and may be unusual in mobility and staining on SDS-PAGE. By modifying the detection method of proteins on SDS-PAGE, we could detect all six subunits encoded by NQR operon in the purified NQR complex. The open reading frame of each subunit was identified from its N-terminal amino acid sequence. << Less

  FEBS Lett. 422:240-242(1998) [PubMed] [EuropePMC]

• Purification and characterization of the recombinant Na(+)-translocating NADH:quinone oxidoreductase from Vibrio cholerae.

  Barquera B., Hellwig P., Zhou W., Morgan J.E., Haese C.C., Gosink K.K., Nilges M., Bruesehoff P.J., Roth A., Lancaster C.R., Gennis R.B.

  The nqr operon from Vibrio cholerae, encoding the entire six-subunit, membrane-associated, Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR), was cloned under the regulation of the P(BAD) promoter. The enzyme was successfully expressed in V. cholerae. To facilitate molecular genetics stu ... >> More

  The nqr operon from Vibrio cholerae, encoding the entire six-subunit, membrane-associated, Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR), was cloned under the regulation of the P(BAD) promoter. The enzyme was successfully expressed in V. cholerae. To facilitate molecular genetics studies of this sodium-pumping enzyme, a host strain of V. cholerae was constructed in which the genomic copy of the nqr operon was deleted. By using a vector containing a six-histidine tag on the carboxy terminus of the NqrF subunit, the last subunit in the operon, the recombinant enzyme was readily purified by affinity chromatography in a highly active form from detergent-solubilized membranes of V. cholerae. The recombinant enzyme has a high specific activity in the presence of sodium. NADH consumption was assessed at a turnover number of 720 electrons per second. When purified using dodecyl maltoside (DM), the isolated enzyme contains approximately one bound ubiquinone, whereas if the detergent LDAO is used instead, the quinone content of the isolated enzyme is negligible. Furthermore, the recombinant enzyme, purified with DM, has a relatively low rate of reaction with O(2) (10-20 s(-1)). In steady state turnover, the isolated, recombinant enzyme exhibits up to 5-fold stimulation by sodium and functions as a primary sodium pump, as reported previously for Na(+)()-NQR from other bacterial sources. When reconstituted into liposomes, the recombinant Na(+)-NQR generates a sodium gradient and a Delta Psi across the membrane. SDS-PAGE resolves all six subunits, two of which, NqrB and NqrC, contain covalently bound flavin. A redox titration of the enzyme, monitored by UV-visible spectroscopy, reveals three n = 2 redox centers and one n = 1 redox center, for which the presence of three flavins and a 2Fe-2S center can account. The V. cholerae Na(+)-NQR is well-suited for structural studies and for the use of molecular genetics techniques in addressing the mechanism by which NADH oxidation is coupled to the pumping of Na(+) across the membrane. << Less

  Biochemistry 41:3781-3789(2002) [PubMed] [EuropePMC]